The objectives were to evaluate the effects of equine chorionic gonadotropin (eCG) supplementation (with or without eCG) and type of ovulatory stimulus (GnRH or ECP) on ovarian follicular dynamics, luteal function, and pregnancies per AI (P/AI) in Holstein cows receiving timed artificial insemination (TAI). On Day 0, 742 cows in a total of 782 breedings, received 2mg of estradiol benzoate (EB) and one intravaginal progesterone (P4) insert (CIDR). On Day 8, the CIDR was removed, and all cows were given PGF2 alpha and assigned to one of four treatments in a 2 x 2 factorial arrangement: (1) CG: GnRH 48 h later; (2) CE: ECP; (3) EG: eCG+GnRH 48 h later; (4) EE: eCG+ECP. There were significant interactions for eCG x ovulatory stimulus and eCG x BCS. Cows in the CG group were less likely (28.9% vs. 33.8%; P<0.05) to become pregnant compared with those in the EG group (odds ratio [OR]=0.28). There were no differences in P/AI between CE and EE cows (30.9% vs. 29.1%; OR=0.85; P=0.56), respectively. Thinner cows not receiving eCG had lower P/AI than thinner cows receiving eCG (15.2% vs. 38.0%; OR=0.20; P<0.01). Treatment with eCG tended to increase serum progestesterone concentrations during the diestrus following synchronized ovulation (P<0.10). However, the treatment used to induce ovulation did not affect CL volume or serum progesterone concentrations. In conclusion, both ECP and GnRH yielded comparable P/AI. However, eCG treatment at CIDR removal increased pregnancy rate in cows induced to ovulate with GnRH and in cows with lower BCS.

Luteal blood flow was studied in heifers by transrectal color-Doppler ultrasound. Data were normalized to the decrease in plasma progesterone to <1 ng/ml (Day 0 or Hour 0). Blood flow in the corpus luteum (CL) was estimated by the percentage of CL area with color flow signals. Systemic prostaglandin F2alpha (PGF) treatment (25 mg; n=4) resulted in a transient increase in CL blood flow during the initial portion of the induced decrease in progesterone. Intrauterine treatment (1 or 2 mg) was done to preclude hypothetical secondary effects of systemic treatment. Heifers were grouped into responders (luteolysis; n=3) and nonresponders (n=5). Blood flow increased transiently in both groups; induction of increased blood flow did not assure the occurrence of luteolysis. A transient increase in CL blood flow was not detected in association with spontaneous luteolysis when examinations were done every 12 h (n=6) or 24 h (n=10). The role of PGF pulses was studied by examinations every hour during a 12-h window each day during expected spontaneous luteolysis. At least one pulse of 13,14-dihydro-15-keto-PGF2alpha (PGFM) was identified in each of six heifers during the luteolytic period (Hours -48 to -1). Blood flow increased (P<0.02) during the 3-h ascending portion of the PGFM pulse, remained elevated for 2 h after the PGFM peak, and then decreased (P<0.03) to baseline. Results supported the hypothesis that CL blood flow increased and decreased with individual PGFM pulses during spontaneous luteolysis.

Reproductive management programs that synchronize ovulation can ovulate a smaller than normal follicle, potentially resulting in inadequate progesterone (P4) concentrations after artificial insemination (AI). Ovulation of the dominant follicle of the first follicular wave with human chorionic gonadotropin (hCG) treatment can produce an accessory corpus luteum and increase circulating P4 concentrations. This manuscript reports the results of 2 separate analyses that evaluated the effect of hCG treatment post-AI on fertility in lactating dairy cows. The first study used meta-analysis to combine the results from 10 different published studies that used hCG treatment on d 4 to 9 post-AI in lactating dairy cows. Overall, pregnancies per artificial insemination (P/AI) were increased 3.0% by hCG treatment post-AI [34% (752/2,213) vs. 37% (808/2,184); Control vs. hCG-treated, respectively]. The second study was a field research trial in which lactating Holstein cows (n=2,979) from 6 commercial dairy herds were stratified by parity and breeding number and then randomly assigned to one of 2 groups: control (no further treatment, n=1,519) or hCG [Chorulon i.m.: 2,000 IU (in 3 of the herds) or 3,300 IU (in 3 herds); n=1,460] on d 5 after a timed AI (ovulation synchronized with Ovsynch, Presynch-Ovsynch, or Double-Ovsynch). In a subset of cows, the hCG profile and P4 changes were determined. Treatment with hCG increased P4 (4.3 vs. 5.3 ng/mL on d 12). Pregnancies per AI were greater in cows treated with hCG (40.8%; 596/1,460) than control (37.3%; 566/1,519) cows. Interestingly, an interaction among treatment and parity was observed; primiparous cows had greater P/AI after hCG (49.7%; 266/535) than controls (39.5%; 215/544). In contrast, older cows receiving hCG (35.7%; 330/925) had similar P/AI to controls (36.0%; 351/975).Thus, targeted use of hCG on d 5 after TAI enhances fertility about 3.0% (based on meta-analysis) to 3.5% (based on our field trial). Surprisingly, this fertility-enhancing effect of hCG was very large in first-lactation cows but not observed in older cows in the field study. Future research is needed to confirm these intriguing results and to determine why older cows did not have improved fertility after hCG treatment.

The luteolytic effects of exogenous prostaglandin F2alpha (PGF) that did and did not simulate natural 13,14-dihydro-15-keto-PGF (PGFM) pulses were studied during mid-diestrus in 42 Holstein heifers. Plasma concentrations of PGF were assessed by assay of PGFM. In experiment 1, a single intrauterine injection of 4.0 mg of PGF into the uterine horn ipsilateral to the corpus luteum resulted in a precipitous progesterone decline, whereas sequential injections of 0.25 or 1.0 mg every 12 h resulted in a stepwise decrease (P < 0.05) following each injection. A progesterone increase occurred during the first 5 min before the luteolytic decrease but only for the 4.0-mg dose. From the results of experiment 2, a 2-h intrauterine infusion of a total of 0.5 mg of PGF was judged to best simulate a natural PGFM pulse. In experiment 3, simulation of sequential pulses at 12-h intervals resulted in a continuous precipitous decrease in progesterone to <1 ng/ml by the beginning of the fourth simulated pulse. In contrast, a single simulated pulse resulted in a 6-h progesterone decrease to a constant concentration for 3 days after treatment, followed by a return to control concentrations. The mean +/- SEM interval between the pretreatment and posttreatment ovulations was shorter (P < 0.05) in the group with sequential simulated pulses (14 +/- 1 day) than in the group with a single pulse (21 +/- 1 day). Results indicated that excessive PGF doses may stimulate nonphysiologic progesterone responses and supported the hypothesis that sequential PGF pulses are required to stimulate natural luteolysis in cattle.

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The hypothesis was tested that estradiol (E2) from the ovarian follicles controls time of luteolysis. Time of luteolysis was evaluated by multiple measures of corpus luteum (CL) structure (area, volume) and function (progesterone [P4], luteal blood flow). The hypothesis for experiment 1 was that repeated ablation of follicles would reduce circulating E2 and delay luteolysis. Heifers were randomly assigned on Day 9 (Day 0 = ovulation) to three groups. All follicles >or=4 mm were ablated on Day 9 (group FA9; n = 6); Days 9-15 (group FA15; n = 6); or Days 9-21 (group FA21; n = 7). As expected, follicular ablation delayed (P < 0.001) the rise in circulating E2 and peak E2 concentrations (FA9, Day 17.6 +/- 0.7; FA15, Day 20.3 +/- 0.3; FA21, Day 24.9 +/- 0.3). Luteolysis (based on each measure) was delayed (P < 0.005) by repeated ablation of follicles, with earlier luteolysis (based on P4 decrease) in FA9 (Day 15.2 +/- 0.8) than FA15 (Day 16.5 +/- 0.4), and a further delay in FA21 (Day 18.3 +/- 0.5). The hypothesis of experiment 2 was that exogenous treatment with E2 would stimulate prostaglandin F(2alpha) (PGF) secretion and prevent the delay in luteolysis associated with follicular ablations. Follicles >or=4 mm were ablated from Day 9 to Day 17 (n = 15). Heifers were treated on Days 13 and 15 with 1.0 mg of estradiol benzoate (FAE2; n = 7) or vehicle (FAV; n = 8). Treatment with E2 induced PGF secretion (detected by PGF metabolite) and induced earlier (P < 0.02) luteolysis in FAE2 than in FAV, whether determined by circulating P4 or by area, volume, or blood flow of CL. In summary, ablation of follicles (>or=4 mm) delayed and treatment with E2 hastened luteolysis in heifers with ablated follicles. Thus, these results are consistent with an essential role for follicle E2 in timing of luteolysis.

Blood collections for characterising 13,14-dihydro-15-keto-PGF2alpha (PGFM) pulses in mares and colour-Doppler examinations for estimating percentage of corpus luteum with blood-flow signals were done hourly for a 24-h session on Day 15 (ovulation = Day 0; n = 13 mares) or during 12-h sessions from Days 12 to 16 (n= 10 mares). Luteolysis was defined as extending from the beginning of a precipitous decrease in progesterone until progesterone was <2 ng mL(-1). Comparisons were made among preluteolysis, luteolysis, and postluteolysis. Greater prostaglandin F2alpha activity (mean PGFM concentration per session) occurred during luteolysis than during preluteolysis and postluteolysis. Statistically-detected PGFM pulses were smaller during preluteolysis with a highly variable interval from the last pulse to the beginning of luteolysis. Either two or three pulses were detected in each 24-h session during luteolysis and postluteolysis, after excluding three of eight sessions with no pulses during postluteolysis. Statistically, 17% of pulses during postluteolysis were prominent outliers. The nadir-to-nadir interval during a pulse (5 h), the peak-to-peak interval between pulses (9 h), and the resulting 4-h gap between pulses were similar during and after luteolysis. The decrease in progesterone encompassed the PGFM pulses, without a detectable fluctuation during a pulse. The percentage of corpus luteum with blood-flow signals did not change during the ascending portion of a PGFM pulse and decreased within 2 or 3 h after the peak, even during preluteolysis. Results indicated that a reported increase in luteal blood flow in heifers during the ascending portion of a PGFM pulse does not occur in mares.

Although somatic cell nuclear transfer (SCNT) is a promising tool, its potential use is hampered by the high mortality rates during the development to term of cloned offspring. Abnormal epigenetic reprogramming of donor nuclei after SCNT is thought to be the main cause of this low efficiency. We hypothesized that chromatin-modifying agents (CMAs) targeting chromatin acetylation and DNA methylation could alter the chromatin configuration and turn them more amenable to reprogramming. Thus, bovine fibroblasts were treated with 5-aza-2'-deoxycytidine (AZA) plus trichostatin (TSA) or hydralazine (HH) plus valproic acid (VPA) whereas, in another trial, cloned bovine zygotes were treated with TSA. The treatment of fibroblasts with either AZA+TSA or HH+VPA increased histone acetylation, but did not affect the level of DNA methylation. However, treatment with HH+VPA decreased cellular viability and proliferation. The use of these cells as nuclear donors showed no positive effect on pre- and postimplantation development. Regarding the treatment of cloned zygotes with TSA, treated one-cell embryos showed an increase in the acetylation patterns, but not in the level of DNA methylation. Moreover, this treatment revealed no positive effect on pre- and postimplantation development. This work provides evidence the treatment of either nuclear donor cells or cloned zygotes with CMAs has no positive effect on pre- and postimplantation development of cloned cattle.